There are a growing number of antineoplastic and antiviral agents such as the nucleoside analogs and dideoxy nucleosides that act as anti-metabolites by inhibiting nucleic acid polymerization, or elongation. Some resistance or ineffectiveness of these agents may be due to an exonuclease activity that removes the analog from the nucleic acid molecule and permits the analog to be replaced with the correct nucleoside.
As an example of such a therapy for treatment of acute myeloblastic leukemia (AML) includes administration of 1-xcex2-D-arabinofuranosylcytosine (araC), an analog of dCTP and potent inhibitor of DNA replication. For a review, see Gilman, et al. (Eds.), The Pharmacological Basis of Therapeutics, Eighth Edition, Pergamon Press; New York (1990), pp. 1230-1232. Despite the well established therapeutic value of araC, the precise mechanism by which cell death is induced is unclear. One possibility is that inhibition of DNA synthesis without concomitant suppression of RNA and protein synthesis leads to xe2x80x9cunbalanced growthxe2x80x9d resulting in increased cell volume and ultimately cell death. In araC treatment, it has been observed that a large number of AML patients are initially refractory to the drug or later develop resistance to araC resulting in failure of therapy in the long term. It is believed that araC resistance arises in part from the relative activities of metabolic enzymes that participate in conversion of araC to araCTP and ultimately to an inactive araUMP. Other factors which may influence araC efficacy include (i) the ability of cells to transport araC, (ii) deoxycytidine kinase deficiency, (iii) increased CTP synthase activity which gives rise to increased intracellular dCTP that may inhibit araC activity, (iv) cytidine deaminase activity, and/or (v) coordinated polymerase/exonuclease activities. Changes in araC structure and/or intracellular concentration relative to analogous compounds may alter affinity of DNA polymerases for araC, thereby resulting in decreased incorporation of the analog into replicating DNA and decreased efficacy of araC chemotherapy regimens.
Thus there exists a need in the art to identify metabolic factors which modulate the ability of chemotherapeutic agents to effect cell killing. Isolation of polypeptides, and their underlying polynucleotide sequences that modulate araC activity would permit the design and identification of therapeutics that regulate the biological activity of the polypeptides and increase efficiency of chemotherapeutic agent at lower doses. Treatment regimens including lower doses of a chemotherapeutic agent may be more easily tolerated in patients, reduce unpleasant side effects, and increase overall efficiency of the treatment program.
The present invention addresses certain shortcomings in the fields of anti-cancer and anti-viral therapies by providing isolated 3xe2x80x2-5xe2x80x2 exonucleases that are not linked to any polymerase activity, and that are shown herein to be involved in decreasing the effectiveness of certain therapeutic compounds, and in particular by providing an isolated human genomic 3xe2x80x2-5xe2x80x2 exonuclease encoding polynucleotide. For example, agents such as nucleoside analogs and chain-terminating dideoxynucleotides, which are used as therapeutic agents against proliferating cells, are removed from a cellular or viral genome by the disclosed exonucleases during treatment, allowing the cell or virus to continue to proliferate. In light of the present disclosure, these isolated exonucleases may be inhibited or even eliminated from a cell containing an anti-proliferative therapeutic agent in order to increase the effectiveness of such an agent.
Disclosed herein are isolated nucleic acid molecules of from about 708 to about 1642 nucleotides in length that include a gene, or the full length complement of a gene, particularly genes that encode a polypeptide, or protein, that includes the amino acid sequence of those sequences designated herein as SEQ ID NO:2, SEQ ID NO:4, SEQ ID 30, SEQ ID NO:32 or SEQ ID NO:34 and conservative variants of these polypeptides. Conservative variants of a polypeptide typically contain an alternative amino acid at one or more sites within the protein. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar size and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isolcucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative variants may also include small deletions or insertions of amino acids, so long as the protein maintains its enzymatic activity.
For example, insertional variants may include fusion proteins such as those used to allow rapid purification of the polypeptide and also may include hybrid proteins containing sequences from other proteins and polypeptides such as homologues of the polypeptide. For example, an insertional variant may include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species. Other insertional variants may include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site, or to aid in chromatographic purification of the polypeptide.
Also disclosed are several regions, hereinafter conserved regions, within these amino acid sequences that are evolutionarily conserved between the human, murine and Drosophila polypeptides or proteins. For example, in one particular region of the polypeptides disclosed herein that includes a contiguous sequence from about amino acids 12 through 25 of SEQ ID NO:2, about amino acids 8 through 21 of SEQ ID NO:4, about amino acids 12 through 25 of SEQ ID NO:30, about amino acids 8 through 21 of SEQ ID NO:32 and about amino acids 18 through 31 of SEQ ID NO:34 is substantially conserved between these three species and may be so among other species as well. Additionally, a second region of conserved amino acid sequence is disclosed herein to be from about amino acid 124 through about 134 of SEQ ID NO:2, from about amino acid 113 or about 117 through about 127 of SEQ ID NO:4, from about amino acid 120 or 124 through about 134 of SEQ ID NO:30, from about amino acid 117 through about 127 of SEQ ID NO:32 and from about amino acid 129 or 133 through about 143 of SEQ ID NO:34 is substantially conserved between these three species and may be so among others. Furthermore, a third region from about amino acids 195 through 205 of SEQ ID NO:2, 188 through 198 of SEQ ID NO:4, 195 through 205 of SEQ ID NO:30, 188 through 198 of SEQ ID NO:32 and 303 through 313 of SEQ ID NO:34 is also conserved among the three species and may be conserved among other species. The disclosed invention also encompasses mutations of the conserved regions which may be conservative in nature, or may be targeted to disrupt or modify enzymatic activity of the polypeptides or may be targeted to disrupt or modify potential interactions with other molecules.
In certain embodiments, the polypeptides or proteins disclosed herein are encoded by the nucleic acid sequences designated herein as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 or the complement, or full length complement of any of these. As used herein the term xe2x80x9ccomplementxe2x80x9d is used to define a second strand of nucleic acid that will hybridize to a first nucleic acid sequence to form a double stranded molecule under highly stringent conditions. Highly stringent conditions are those that allow hybridization between two nucleic acid sequences with a high degree of homology, but precludes hybridization of random sequences. For example, hybridization at low temperature and/or high ionic strength is termed low stringency and hybridization at high temperature and/or low ionic strength is termed high stringency. In a general sense, a low stringency hybridization may include conditions of 0.15 M to 0.9 M NaCl at a temperature range of 20xc2x0 C. to 50xc2x0 C. High stringency may generally include conditions of 0.02 M to 0.15 M NaCl at a temperature range of 50xc2x0 C. to 70xc2x0 C. Preferred nucleic acid segments as disclosed herein are those that hybridize to the nucleic acid sequences designated herein as SEQ ID NOS:1, 3, 29, 31 or 33 under conditions including hybridization at 50xc2x0 C. in 1xc3x97SSC, and washing at 65xc2x0 C. in 0.1xc3x97SSC. As known in the art, 1xc3x97SSC is a solution containing about 8.76 grams/liter NaCl and about 4.41 grams/liter sodium citrate. The temperature and ionic strength of a desired stringency are understood to be applicable to particular probe lengths, to the length and base content of the sequences and to the presence of formamide, tetramethylammonium chloride or other solvents in the hybridization mixture. It is also understood that these ranges are mentioned by way of example only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to positive and negative controls. To hybridize is understood to mean the forming of a double stranded molecule or a molecule with substantial double stranded nature.
Equations have been derived to relate duplex formation to the major variables of temperature, salt concentration, nucleic acid strand length and composition, and formamide concentration.
Eg:
Tm=81.5xe2x88x9216.6(log[Na+])+0.41(%GC)xe2x88x92600/Nxe2x80x83xe2x80x831.
(Tm=temperature for duplex to half denature; N=chain length
xe2x80x83Tm=81.5xe2x88x9216.6(log[Na+]+0.41(%GC)xe2x88x920.63(% formamide)xe2x88x92600/Nxe2x80x83xe2x80x832.
One can thus predict whether complementary strands will exist in double-stranded or single-stranded form under a given set of conditions, and can determine high stringency conditions based on knowledge of the nucleotide sequences.
It is understood in the art that a nucleic acid sequence will hybridize with a complementary nucleic acid sequence under high stringency conditions even though some mismatches may be present. Such closely matched, but not perfectly complementary sequences are also encompassed by the present invention. For example, differences may occur through genetic code degeneracy, or by naturally occurring or man made mutations and such mismatched sequences would still be encompassed by the present disclosure. A complement may also be described, therefore, as a fragment of DNA (nucleic acid segment) or a synthesized single stranded oligomer that may contain small mismatches or gaps when hybridized to its complement, but that is able to hybridize to the complementary DNA under high stringency conditions. The full length complement is understood to indicate that the two molecules hybridize along the full length of the gene or complementary region. For example the full length complement of a gene would be a complementary molecule that is complementary along the entire gene rather than complementary to only a small portion of the gene. It is also understood that a nucleic acid strand that includes the full length complement of a gene may also contain extraneous nucleotides flanking the complementary region, or linked to either end of the complementary region and such strands would still be defined as the full length complement of the gene. Furthermore, it is understood that the portions of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:29, SEQ ID NO:31 and SEQ ID NO:33 that encode the conserved regions would be particularly useful in the identification of other exonucleases either intra- or inter-species searches. The present invention also encompasses the use of degenerate probes targeted to said conserved regions.
The nucleic acid molecules disclosed herein are, in certain embodiments, operatively linked to a promoter, and may be operatively linked to the autogenous promoter for that gene or to a heterologous promoter, and may be linked to any appropriate promoter known in the art that is appropriate for the particular application. For example, certain promoters may be chosen for expression in a particular type of cell, or for high expression, or even for inducible expression of the gene of interest. The selection of such promoters is well known and routine in the art, and a comprehensive list of all available promoters is available from various sources to those in the art. In certain embodiments the gene will be operatively linked to its own (autogenous) promoter, either as the promoter is present in a cell, or with nucleic acid sequence added to, or removed from the nucleic acid molecule between the promoter region and the translational start site. A preferred promoter for use in the present invention may be, for example, a promoter contained in the nucleic acid sequence designated herein as SEQ ID NO: 1, in particular those sequences from about base 9 to about base 59, or from about base 519 to about base 569 of SEQ ID NO:1. A gene as disclosed herein may also be linked to various markers known in the art to monitor transformation efficiency or to otherwise detect the presence of the gene. Such markers are also routine and known in the art.
The nucleic acids of the present disclosure may also be contained in a vector. A vector used in the practice of the invention may be a plasmid, a viral vector, and also may be an expression vector that directs expression of the disclosed genes in an appropriate host cell. Vectors as described herein may be compatible with certain host cells such as bacterial cells, yeast, plant, animal, or even mammalian cells. Certain aspects of the disclosure may also include vectors contained in host cells.
In certain embodiments, the present disclosure encompasses compositions containing purified or partially purified proteins or polypeptides. Such partially purified polypeptides having a 3xe2x80x2-5xe2x80x2 exonuclease activity, include those having or including the amino acid sequences designated herein as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:34 or a conservative variant of any thereof. A polypeptide as disclosed herein may be a naturally occurring protein that is isolated from a cell, such as a mammalian cell or even a mouse or human cell, using chromatographic or other techniques as disclosed herein or known in the art. Such techniques generally include isolation of a particular fraction of a cell culture, such as the aqueous fraction, for example, and a protein precipitation in the presence of an ammonium salt, such as ammonium sulfate.
Polypeptides as disclosed herein may also be recombinant proteins or polypeptides expressed from a manmade vector or isolated gene. Such recombinant proteins may also be isolated from a cell culture as described for the naturally occurring proteins, but are often xe2x80x9coverexpressedxe2x80x9d at a higher level than normal.
As such, a method for producing a polypeptide having 3xe2x80x2-5-exonuclease activity is also disclosed herein. Such a method may include obtaining a nucleic acid molecule including a gene encoding a polypeptide including the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:34 or a conservative variant of any thereof, operatively linked to a promoter sequence; transferring the nucleic acid molecule into a host cell; and growing the host cell under conditions effective to express the gene. In certain embodiments, the method may further include isolating the polypeptide from a host cell or from the medium of its growth. It is also understood that in certain embodiments a recombinantly produced protein may be used in the intracellular compartment where it is expressed, in a candidate screening assay for example, and such methods would not require isolation of the protein product. Proteins and polypeptides of the invention may be produced in any appropriate cell, including but not limited to, bacterial cells and eukaryotic cells such as mammalian cells.
The present disclosure also encompasses antibodies specifically immunoreactive with a polypeptide that includes the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:34 and more particularly, antibodies with specific reactivity to the conserved regions described above. Antibodies may be polyclonal or monoclonal antibodies, although monoclonal antibodies are preferred for certain embodiments, and may also include anti-idiotype antibodies specifically immunoreactive with the disclosed antibodies.
An aspect of the present disclosure is a method of identifying an effector of a 3xe2x80x2-5xe2x80x2 exonuclease activity. This method includes obtaining a candidate substance; contacting a 3xe2x80x2-5xe2x80x2 exonuclease polypeptide composition with a substrate in the presence and absence of the candidate substance; and detecting 3xe2x80x2-5xe2x80x2 exonuclease activity in the presence and absence of the candidate substance; wherein a change in activity of the exonuclease in the presence of the candidate substance is indicative of an effector of 3xe2x80x2-5xe2x80x2 exonuclease activity. Also encompassed herein are effectors of 3xe2x80x2-5xe2x80x2 exonuclease activity identified by this method, and pharmaceutical compositions including such an effector. An effector of exonuclease activity may be an activator or an inhibitor of the enzymatic activity, or even of the expression of the protein in a cell. Furthermore, an effector of 3xe2x80x2-5xe2x80x2 exonuclease activity might be a variant form of an exonuclease peptide itself with or without exonuclease activity.
A method of identifying an inhibitor of 3xe2x80x2-5xe2x80x2 exonuclease activity may include obtaining a candidate substance; growing a cell culture in the presence of a nucleoside analog that is incorporated into a nucleic acid molecule and inhibits polymerization of the molecule when incorporated therein, wherein the cells express a 3xe2x80x2-5-exonuclease activity; contacting the cell culture with the candidate substance; growing an identical cell culture that is not contacted with the candidate substance; and comparing the cell growth in the presence and absence of the candidate substance; wherein a decrease in cell growth in the presence of the candidate substance is indicative of an inhibitor of 3xe2x80x2-5xe2x80x2 exonuclease activity. It is an aspect of the present disclosure that an effector of the 3xe2x80x2-5xe2x80x2 exonuclease activity may either interact with a 3xe2x80x2-5xe2x80x2 exonuclease protein and inhibit its activity through direct contact, or a substance may inhibit the expression of a gene encoding the 3xe2x80x2-5xe2x80x2 exonuclease protein through interaction with the promoter or other control sequence, or even with a portion of the coding sequence of the gene, such as an antisense molecule, for example. As such, both an effector of the protein product, and an effector of genetic expression of the protein product are aspects of the present disclosure, and would be useful in the practice of the present invention.
A screening assay as described herein may include obtaining a candidate substance, which can come from any source. For example, it is proposed that compounds isolated from natural sources such as fungal extracts, plant extracts, bacterial extracts, higher eukaryotic cell extracts, or even extracts from animal sources, or marine, forest or soil samples, may be assayed for the presence of potentially useful pharmaceutical agents. In addition, man made or synthetic substances which would include, but are not limited to, nucleic acid analogs, peptides, polypeptides or other compounds designed de novo based on the predicted protein structure of the exonuclease enzyme, may also be screened for possible use as pharmaceutical agents, or as agents to be used in combination with other pharmaceutical agents. It is also understood that antibodies and other isolated or purified, but naturally occurring compounds, could be screened by this process. The active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.
The present disclosure also includes methods of inhibiting the replication of a nucleic acid molecule in a cell that expresses a 3xe2x80x2-5xe2x80x2 exonuclease activity comprising contacting said cell with a nucleic acid polymerization inhibitor such as a nucleoside analog or a dideoxy nucleotide, and further contacting the cell with an inhibitor of the 3xe2x80x2-5xe2x80x2 exonuclease activity. This method may preferably be practiced in any type of cell, including, but not limited to, a mammalian cell, a human cell, or even a human cancer cell. The method is particularly advantageous when applied to a proliferating tumor or cancer cell, or a virally infected cell, such as a mammalian cell infected with a virus, and including T-cells and monocyte/macrophage. Viruses would include, but not be limited to, retroviruses including HIV, herpes simplex viruses (1 and 2), Epstein-Barr viruses, varicella-zoster viruses, influenza viruses, Lassa fever, infectious hepatitis, dengue fever, measles, respiratory syncytial viruses, vaccinia viruses, and cytomegaloviruses, for example. As a part of this method, one may include any dideoxynucleotide, such as ddATP, ddGTP, ddCTP, ddUTP, ddTTP or even ddITP, and may also include nucleoside analogs, and compounds that are converted to nucleoside analogs in the cell. Such drugs would include cytarabine, fluorouracil, mercaptopurine, thioguanine, acyclovir, didanosine, ganciclovir sodium, idoxuridine, ribavirin, trifluridine, zalcitabine, azacitidine, and zidovudine, for example.
Also disclosed are methods of identifying a compound as a specific binding partner of the exonuclease polypeptide. In a preferred method, the specific binding partner modulates activity of the exonuclease polypeptide. In a most preferred embodiment, the methods of the invention identify compounds that inhibit biological activity of the exonuclease polypeptide. It is contemplated that compounds that interact with active site amino acids, such as amino acids 2 through 17, 111 through 125, or 181 through 196 of SEQ ID NO:2, or amino acids 8 through 24, 114 through 128, or 184 through 199 of SEQ ID NO:4 may be particularly useful. In addition, it is contemplated that compounds which interact with the conserved regions, individually or in combination, of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:34 would also be particularly useful.
The invention also provides methods to identify an inhibitor compound of an exonuclease biological activity comprising the steps of a) contacting the exonuclease polypeptide encoded by a polynucleotide of the invention with a substrate in the presence and absence of a test compound; b) comparing biological activity of the exonuclease polypeptide in the presence and absence of the test compound; and c) identifying the test compound as an inhibitor compound when biological activity of the exonuclease polypeptide is decreased in the presence of the test compound. Also provided are inhibitors identified by the method and pharmaceutical compositions comprising an inhibitor identified by the method of the invention.
It is a further aspect of the invention that one may obtain a genetic construct containing a promoter region, or a control region of the 3xe2x80x2-5xe2x80x2 exonuclease, and particularly a human TREX1 gene promoter, such as a promoter region contained in SEQ ID NO:1, or another promoter that may be isolated from the human or other animal genome using the sequence, or fragments of the sequence disclosed herein as SEQ ID NO:1. Such a construct will contain an encoded gene operatively linked to the promoter region such that the gene is under the transcriptional control of the promoter region. In certain embodiments the encoded gene will be a gene encoding a 3xe2x80x2-5xe2x80x2 exonuclease and in certain embodiments a gene encoding a reporter gene may be included. Such reporter genes would include, but would not be limited to a luciferase gene, an antibiotic resistance marker, or an essential metabolic gene, a xcex2-galactosidase gene from E. coli or a chloramphenicol acetyltransferase gene, for example. In this embodiment, the reporter gene would be expressed in the presence and absence of a candidate substance as in the previously described screening assays. A change in level of the reporter gene product in the presence of the candidate substance relative to the level in the absence of the candidate substance would indicate an effector of 3xe2x80x2-5xe2x80x2 exonuclease expression.
An increase in reporter gene activity over a control would indicate an activating substance and a decrease in activity over the control would indicate an inhibitor. It is understood that, in the assays described herein, the inhibition of 3xe2x80x2-5xe2x80x2 exonuclease activity in a cell could occur at any level of the expression of 3xe2x80x2-5xe2x80x2 exonuclease activity, including gene transcription, RNA processing, mRNA translation, post translational modification and even protein transport or at any other level that would have the overall effect of activation or inhibition of 3xe2x80x2-5xe2x80x2 exonuclease activity, and that the methods described and claimed would include effectors at any of these, or any other level of protein expression. Preferred cells to be used in the assay would be Chinese hamster ovary cells (CHO) for example, however, any cells which are capable of expressing a 3xe2x80x2-5xe2x80x2 exonuclease, or a reporter gene as described herein would be acceptable and would be encompassed by the present claimed invention. Examples of other cell types include MDCK, CaCo2, BHK, COS AND 293 cells, for example.
The invention further provides methods for increasing incorporation of a nucleotide analog into replicating DNA in a cell comprising the steps of, a) contacting the cell with a nucleotide analog, and b) contacting the cell with an inhibitor of an exonuclease polypeptide activity encoded by a polynucleotide as disclosed herein. In a preferred method, the exonuclease is selected from the group consisting of TREX1h, TREX2h, TREX1m, or TREX2m. The inhibitor may be a substrate analog, an antibody, an antisense molecule, or another exonuclease peptide with or without exonuclease activity, or an inhibitor of either gene expression or enzymatic activity. An inhibitor may be included in a pharmaceutical composition including a nucleotide analog such as araC, or chain terminating nucleotide such as a dideoxy nucleotide, or it may be administered separately.